Oseltamivir Resistance Research Articles

Frequency and distribution of H1N1 influenza A viruses with oseltamivir-resistant mutations worldwide before and after the 2009 pandemic.

H1N1 influenza has brought serious threats to people's health and a high socioeconomic burden to society. Oseltamivir, a kind of neuraminidase (NA) inhibitor, is the second-generation specific drug that is broadly used currently. However, H1N1 influenza viruses have exhibited oseltamivir resistance in the past decades, which might be a hidden danger. To understand the frequency and distribution laws of oseltamivir-resistant viruses, we conducted a thorough and deep analysis of the available NA protein sequences of H1N1 influenza viruses worldwide from 1918 to 2020. The differences and similarities before and after 2009 were also considered since the dominant viruses changed in this period. Results showed that 3.76% of H1N1 viruses harbored oseltamivir resistance currently. Among various significative mutations, H274Y had the highest frequency of 3.30%, while the frequencies of the other mutations were far below this whether before or after 2009. The oseltamivir resistance was mainly found in three hosts, humans, swine, and avian. Different mutation sites could exhibit different distributions in each host. Our results showed that the resistance level reached a peak during the 2007-2008 influenza season and then quickly decreased in 2009. The resistance also displayed a global distribution. The densely populated countries usually had a high resistance level. However, frequent significative mutations were also found in some small countries. Our findings indicated the necessity of monitoring oseltamivir resistance around the world. The study could provide a unique perspective towardthe cognition of viruses and facilitate the future study of both pandemic and drug development.

Genetic Analysis of Influenza A/H1N1pdm Strains Isolated in Bangladesh in Early 2020.

Influenza is one of the most common respiratory virus infections. We analyzed hemagglutinin (HA) and neuraminidase (NA) gene segments of viruses isolated from influenza patients who visited Evercare Hospital Dhaka, Bangladesh, in early 2020 immediately before the coronavirus disease 2019 (COVID-19) pandemic. All of them were influenza virus type A (IAV) H1N1pdm. Sequence analysis of the HA segments of the virus strains isolated from the clinical specimens and the subsequent phylogenic analyses of the obtained sequences revealed that all of the H1N1pdm recent subclades 6B.1A5A + 187V/A, 6B.1A5A + 156K, and 6B.1A5A + 156K with K209M were already present in Bangladesh in January 2020. Molecular clock analysis results suggested that the subclade 6B.1A5A + 156K emerged in Denmark, Australia, or the United States in July 2019, while subclades 6B.1A5A + 187V/A and 6B.1A5A + 156K with K209M emerged in East Asia in April and September 2019, respectively. On the other hand, sequence analysis of NA segments showed that the viruses lacked the H275Y mutation that confers oseltamivir resistance. Since the number of influenza cases in Bangladesh is usually small between November and January, these results indicated that the IAV H1N1pdm had spread extremely rapidly without acquiring oseltamivir resistance during a time of active international flow of people before the COVID-19 pandemic.

Identification of a Permissive Secondary Mutation That Restores the Enzymatic Activity of Oseltamivir Resistance Mutation H275Y.

Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.

Susceptibility of epidemic viruses to neuraminidase inhibitors and treatment-emergent resistance in the Japanese 2019-20 influenza season

To investigate the susceptibility of epidemic influenza viruses to neuraminidase inhibitors (NAIs) and the emergence of resistant viruses after treatment, a prospective, observational study was done in the 2019-20 Japanese influenza season. Influenza viruses were isolated before and twice after treatment, the first at day 5 and the second at day 10. The 50% inhibitory concentrations (IC50s) to oseltamivir, zanamivir, peramivir, and laninamivir were measured and compared with those of 2010-11 to 2018-19 seasons. NA amino acid sequences were analyzed by next generation sequencing (NGS). The IC50 geometric means of the NAIs for 128 A(H1N1)pdm09, 2 A(H3N2), and 33 B were comparable to those of the previous seasons. Only 2 (1.6%) A(H1N1)pdm09 with significantly high IC50 to oseltamivir were found pretreatment. No A(H3N2) or B had resistance. Treatment-emergent oseltamivir resistance was found in 2 among 33 oseltamivir-treated A(H1N1)pdm09, only at the second follow-up. The NGS indicated a rapid increase in the proportion of H275Y to wild type, from 0% to almost 100% between days 5 and 10. These results suggest the continued effectiveness of these NAIs for epidemic influenza in Japan. Treatment-emergent resistant H275Y A(H1N1)pdm09 viruses were detected after oseltamivir treatment, rapidly replacing the wild type.

Drug resistance of influenza A (H1N1) pdm09 virus in Hebei, 2017–2020

Objective To analyze the resistance of influenza A (H1N1) pdm09 virus to neuraminidase inhibitors (NAIs) in Hebei province during 2017–2020 and provide evidence for the prevention and control of influenza． Methods A total of 37 strains of influenza A (H1N1) pdm09 virus isolated in Hebei during 2017–2020 were selected for oseltamivir and zanamivi resistance tests with fluorescence radiation assay. A total of 38 strains of influenza A (H1N1) pdm09 virus were selected for neuraminidase (NA) sequencing analysis. Results The median of half maximal inhibitory concentration (IC50) of oseltamivir to strain A/Hebeixinhua/SWL1106/2017 was 30.98 nmol/L. To 36 virus strains, the average IC50 of oseltamivir was of 0.46 nmol/L (ranged 0.07–1.14 nmol/L) while the average IC50 of zanamivi was 0.27 nmol/L (ranged 0.07–0.85 nmol/L). Sequence analysis showed that 1 strain had H275Y mutation in amino acid of the NA gene, suggesting that the strain was oseltamivir resistant. Conclusion The majority of influenza A (H1N1) pdm09 virus strains isolated in Hebei during 2017–2020 were sensitive to NAIs.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/μL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

Nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission, United Kingdom, 2018/19 influenza season.

BackgroundInfluenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions.AimTo evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings.MethodsWe conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data.ResultsCompared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome.ConclusionNanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.

Investigation of genetic variation: Neuraminidase gene of influenza Avirus H1N1/pdm09, Shiraz, Iran (2015-2016).

Oseltamivir and antiviral agents are frequently used for the prevention and treatment of influenza infection. However, resistance to oseltamivir has been reported globally due to a mutation in the Influenza virus neuraminidase gene. Such resistance will be detected by genotyping and phenotyping studies of viral isolates. The recent study aimed to determine the genetic mutation of neuraminidase gene in influenza A (H1N1) viruses isolated from children referred to Shiraz tertiary hospitals during 1 year (2015-2016) with influenza-like symptoms. A total of 300 patients were registered and throat samples were taken. The throat swabs were used for viral RNA extraction. Detection of influenza A (H1N1) was performed using the one-step real-time polymerase chain reaction (qRT-PCR) method. From positive isolates for H1N1, 51 random samples were evaluated for neuraminidase gene mutation with the nested PCR-sequencing method. Of 300 cases, 102 (34%) isolates were detected as influenza A (H1N1) pdm09. Based on sequencing results, 2 of the 44 sequenced isolates exhibited H275Y substitution, which presented oseltamivir resistance. In comparison with reference strain, the phylogenetic analysis of sequenced isolates was classified in genogroup 6B. While this result is the first report of emerging oseltamivir-resistant in the southwest of Iran, it is highly recommended to perform these evaluations on the different geographical regions in any prevalence area to plan treatment strategies for influenza.

Source of oseltamivir resistance due to single E276D, R292K, and double E276D/R292K mutations in H10N4 influenza neuraminidase

Influenza is a respiratory contagious disease infecting people all around the globe. The Center for Disease Control and Prevention had estimated that from 1 October 2018 to 4 May 2019, there have been about 40 million flu patients, in which about 50 thousand cases have died. Many subtypes of influenza have developed drug resistance due to high mutation rates. Neuraminidase (NA) is the glycoprotein on the virus particle surface. Its function is to cleave the glycosidic bond with the sialic acid, leading the new virus to be able to infect other uninfected cells. Therefore, it is a promising protein target for drug design and development. The E276D and R292K NA mutations in the H10N4 influenza virus have been reported to cause drug resistance. In this study, molecular dynamics simulations and free energy calculations were applied to study the source of oseltamivir resistance in E276D, R292K, and E276D/R292K NA strains. The obtained results suggested that all studied mutants reduced the number of contact atoms, interaction energies, H-bonds, per-residue interaction energies, and total binding free energies towards the oseltamivir binding, resulting in a lower susceptibility. Only the interactions at residues 118, 119, and 371 were maintained in stabilizing the oseltamivir. The opening at 150- and 430-loops in E276D and double mutations caused the drug unbinding from the active site, increasing water accessibility into the binding pocket of NA enzyme.

Baloxavir treatment of oseltamivir-resistant influenza A/H1pdm09 in two immunocompromised patients.

Few treatment options are available for oseltamivir-resistant influenza. It has been proposed that baloxavir can be effective in this setting due to its distinct mechanism of action but clinical experience is lacking for immunocompromised patients. We report two such cases treated with baloxavir after failure of oseltamivir and detection of oseltamivir resistance mutations. Baloxavir/zanamivir combination therapy was effective in one patient, but persistent viral shedding was noted with baloxavir monotherapy in the other patient.

Intravenous Zanamivir: A Viable Option for Critically Ill Patients With Influenza.

To review the pharmacology, clinical trial data, and clinical implications for the intravenous formulation of zanamivir. MEDLINE, PubMed, EMBASE, and Google Scholar were searched during November 2019 to July 2020. Search terms zanamivir and neuraminidase inhibitor were used. All human trials and major reports from compassionate use programs with the intravenous zanamivir (IVZ) formulation were assessed and reviewed here. IVZ was found to be similar but not superior to oral oseltamivir in hospitalized patients when studied in populations with very low baseline oseltamivir resistance. IVZ provides an effective alternative for critically ill patients when oral antiviral therapy is not preferred or when oseltamivir resistance is increased. IVZ was recently authorized for use by the European Medicines Agency, and it is eligible for consideration in emergency use protocols and US stockpile inclusion. It will be of particular interest in critically ill patients especially during influenza seasons with appreciable oseltamivir and peramivir resistance. The available information suggests that the intravenous formulation of zanamivir offers a viable alternative treatment for critically ill patients with influenza, especially when resistance to other agents is present.

Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity.

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.

Genetic Sequencing of Influenza A (H1N1) pdm09 Isolates from South India, Collected between 2011 and 2015 to Detect Mutations Affecting Virulence and Resistance to Oseltamivir

AbstractBackground: Influenza A viruses evolve continuously and the two surface antigens, hemagglutinin (HA) and neuraminidase (NA) have been the target proteins for research as they are vital components in determining the virulence, immune effectiveness, pathogenicity, transmission and resistance. Methods: Both HA and NA (partial genes) of 45 pandemic influenza A(H1N1)pdm09 isolates were sequenced. Phylogenetic analysis was performed with reference to representative global isolates retrieved from Influenza Virus Resource (IVR), GISAID EpiFluTM and GenBank and evolutionary analyses. Nucleotide and amino acid sequences were aligned using ClustalW/Clustal Omega/MEGA version 6 with reference to vaccine strain (A/California/07/2009). Results: All the isolates clustered along with the clade 7 virus, irrespective of the year of isolation. The study isolates exhibited 98.5% and 98.8% nucleotide homology to the reference strain A/California/07/2009(H1N1) for HA and NA, respectively. Overall, there was limited genetic diversity observed over a period of 3 years (2012-2015). Two samples collected from expired patients had D239N (D222G or D225G) mutation in HA. This mutation which is associated with dual-binding specificity of the virus has been well-correlated with severe disease outcomes. All the study isolates possessed H274 residue and 7 strains had N295S, the next most common mutation found in oseltamivir-resistant variants. Conclusion: In this study, although H274Y mutation associated with oseltamivir resistance has not been noted, significant mutations have been noted in both HA and NA genes including D239N, N295S, V106I, Q136K, N248D, V267A. In both HA and NA gene analysis, multiple mutations were found more in 2015 strains when compared to 2012 strains. Hence such accumulation of mutations has to be monitored continuously to determine the efficacy of annual flu vaccines and anti-influenza drugs.

Combinatory Treatment with Oseltamivir and Itraconazole Targeting Both Virus and Host Factors in Influenza A Virus Infection.

Influenza virus infections and their associated morbidity and mortality are a major threat to global health. Vaccination is an effective influenza prevention measure; however, the effectiveness is challenged by the rapid changes in the influenza virus genome leading to viral adaptation. Emerging viral resistance to the neuraminidase inhibitor oseltamivir limits the treatment of acute influenza infections. Targeting influenza virus-host interactions is a new and emerging field, and therapies based on the combination of virus- and host-directed drugs might significantly improve treatment success. We therefore assessed the combined treatment with oseltamivir and the repurposed antifungal drug itraconazole on infection of polarized broncho-epithelial Calu-3 cells with pdm09 or Panama influenza A virus strains. We detected significantly stronger antiviral activities in the combined treatment compared to monotherapy with oseltamivir, permitting lower concentrations of the drug than required for the single treatments. Bliss independence drug interaction analysis indicated that both drugs acted independently of each other. The additional antiviral effect of itraconazole might safeguard patients infected with influenza virus strains with heightened oseltamivir resistance.

Swine influenza viruses and pandemic H1N1-2009 infection in pigs, Myanmar.

Swine influenza virus (SIV) causes respiratory diseases in pigs and has impacts on both animal and human health. In this study, we conducted swine influenza surveillance in pig farms in the Yangon and Bago regions, Myanmar, during 2017-2019. Nasal swabs (n=500) were collected from pigs in 10 swine farms. Our results showed that 11 out of 100 pooled samples (11%) were positive for influenza A virus (IAV) by real-time RT-PCR. Five SIVs could be isolated and could be subtyped as SIV-H1N1 (n=4) or SIV-H3N2 (n=1). The viruses were further characterized by whole-genome sequencing and classified as pdmH1N1-2009 (n=3), reassortant H1N1 (n=1) or reassortant H3N2 (n=1). Phylogenetic analysis of Myanmar SIVs showed that all genes of the three SIV-H1N1 (pdmH1N1-2009) were clustered with viruses of the pdm/09 lineage. For one SIV-H1N1 (rH1N1), the HA1 gene was clustered with those of endemic SIVs of the classical swine lineage, and seven genes were clustered with those of viruses of the pdm/09 lineage. For SIV-H3N2 (rH3N2), the HA3 and NA2 genes were clustered with those of endemic SIVs of the human-like swine lineage, while six internal genes were clustered with those of viruses of the pdm/09 lineage. Genetic analysis indicated that all the Myanmar SIVs possessed amino acids that favour binding to the human receptor. All the Myanmar SIVs contained amino acids related to amantadine resistance but not oseltamivir resistance. Notably, the pdmH1N1-2009 virus might have been circulating in the Myanmar pig population for a period of time after pdmH1N1-2009 outbreaks in humans. Then, reassortment between endemic SIV-H1N1 or SIV-H3N2 and pdmH1N1-2009 in pig farms in Myanmar could have occurred. Our findings ascertained the genetic diversity of SIVs, especially pdmH1N1-2009, in the pig population in Myanmar, with zoonotic and reverse zoonotic potentials.

Baloxavir for the treatment of Influenza in allogeneic hematopoietic stem cell transplant recipients previously treated with oseltamivir

Seasonal influenza causes significant morbidity and mortality in allogeneic stem cell transplant (SCT) recipients. In this population, influenza virus can replicate for prolonged periods, despite neuraminidase inhibitor treatment, leading to resistance and treatment failure. Baloxavir targets the influenza polymerase and may be an effective treatment option in these patients. We used baloxavir to treat five allogeneic SCT recipients that were still symptomatic and shedding influenza virus after completing one or more treatment courses of oseltamivir and characterized the viral isolates before and during treatment. Two patients were infected with influenza A/H1pdm09 carrying a neuraminidase variant (H275Y) linked to oseltamivir resistance. Both these two patients were successfully treated with baloxavir. Of the three patients infected with wild-type influenza virus, two cleared the virus after baloxavir treatment, while the third patient developed the polymerase I38T variant linked to baloxavir resistance. Our data suggest that baloxavir treatment can be effective in treating neuraminidase inhibitor-resistant influenza in profoundly immunocompromised patients. Randomized clinical trials are needed to define the role of baloxavir alone and combined with oseltamivir for the treatment of influenza in SCT recipients and other immunocompromised populations.

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

Source of oseltamivir resistance due to single E119D and double E119D/H274Y mutations in pdm09H1N1 influenza neuraminidase.

Influenza epidemics are responsible for an average of 3-5 millions of severe cases and up to 500,000 deaths around the world. One of flu pandemic types is influenza A(H1N1)pdm09 virus (pdm09H1N1). Oseltamivir is the antiviral drug used to treat influenza targeting at neuraminidase (NA) located on the viral surface. Influenza virus undergoes high mutation rates and leads to drug resistance, and thus the development of more efficient drugs is required. In the present study, all-atom molecular dynamics simulations were applied to understand the oseltamivir resistance caused by the single E119D and double E119D/H274Y mutations on NA. The obtained results in terms of binding free energy and intermolecular interactions in the ligand-protein interface showed that the oseltamivir could not be well accommodated in the binding pocket of both NA mutants and the 150-loop moves out from oseltamivir as an "open" state. A greater number of water molecules accessible to the binding pocket could disrupt the oseltamivir binding with NA target as seen be high mobility of oseltamivir at the active site. Additionally, our finding could guide to the design and development of novel NA inhibitor drugs.

Characterization of viral genomic mutations in novel influenza A (H7N9)-infected patients: the association between oseltamivir-resistant variants and viral shedding duration.

Since February 2013, human infections with the novel influenza A H7N9 virus have occurred in eastern China. It is important to detect mutations in viral genes and analyze the clinical features of patients and viral shedding duration related to neuraminidase inhibitor (NAI) resistance. We collected clinical specimens from 31 hospitalized H7N9 patients and sequenced NA, PB2, HA, and M gene fragments. Of the 31 identified patients, 7 (22.6%) carried the R292K substitution in NA, 30 (96.8%), 3 (9.7%), and 5 (16.1%) carried E627K, Q591K, and D701N mutations in PB2, respectively, and 2 (6.5%) carried both E627K and D701N mutations in PB2. All 26 identified patients harbored Q226L mutations and possessed only a single arginine (R) at cleavage sites in the HA and a S31N mutation in M2. Among 7 NA-R292K mutated patients, 3 died and 4 were discharged. There was no significant difference in the days that patients started oseltamivir treatment after symptom onset between NA-R292K mutant and NA-R292 wild-type patients (median days, 7 vs 6, P = 0.374). NA-R292K mutant patients had a significantly longer duration of viral shedding than NA-R292 wild-type patients after oseltamivir treatment (median days, 10 vs 5, P = 0.022). The mutation of R292K in NA conferring the potential ability of oseltamivir resistance resulted in prolonged viral duration and poor outcome and should be taken into consideration in the clinical management of infected patients.Electronic supplementary materialThe online version of this article (10.1007/s11262-019-01678-8) contains supplementary material, which is available to authorized users.

Genetic Characterisation of Influenza A(H1N1)pdm09 Viruses Circulating in Assam, Northeast India during 2009–2015

AbstractIntroduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1) pdm09 viruses detected in Assam of Northeast India during 2009–2015. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation ‘N248D’ which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.